Cholesterol Sensitivity and Mechanisms of MSC Responses to 3D Substrate Rigidity

胆固醇敏感性和 MSC 对 3D 基质刚性的响应机制

• 批准号: 9240628
• 负责人: ROCKY S TUAN
• 金额: $ 33.83万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240628
• 关键词: 3-Dimensional; Adherence; Adhesions; Adhesives; Adopted; Adult; Affect; Atomic Force Microscopy; Attention; Behavior; Biological; Biological Assay; Bone Marrow; Brain; Caveolae; Caveolins; Cell Adhesion; Cell Differentiation process; Cell Fractionation; Cell Shape; Cell membrane; Cells; Cellular Morphology; Characteristics; Cholesterol; Cholesterol Homeostasis; Clinical; Cytoskeletal Modeling; Electron Microscopy; Endocytosis; Event; F-Actin; Flow Cytometry; Fluorescence Polarization; Focal Adhesions; Future; Gelatin; Gene Expression Profiling; Goals; Health; Homeostasis; Human; Hyaluronan; Hydrogels; Hypersensitivity; Immunofluorescence Immunologic; Integrins; Knowledge; Ligands; Mediating; Membrane; Membrane Fluidity; Membrane Microdomains; Mesenchymal Stem Cells; Methacrylates; Morphology; Neurons; Osteoblasts; Osteocalcin; Osteogenesis; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phenotype; Plasticizers; Population; Property; Reverse Transcriptase Polymerase Chain Reaction; Role; Rupture; Seeds; Shapes; Signal Pathway; Signal Transduction; Small Interfering RNA; Sucrose; Supplementation; Suspensions; System; Testing; Time; Tissue Engineering; Tissues; Western Blotting; base; caveolin 1; cell behavior; chemical property; design; ethylene dimethacrylate; in vivo; in vivo regeneration; inhibitor/antagonist; knock-down; mechanical properties; mechanotransduction; osteogenic; physical property; regenerative therapy; response; scaffold; soft tissue; tissue regeneration

ABSTRACT We propose a mechanistic study of the biological responses of adult human bone marrow-derived mesenchymal stem cells (MSCs) to 3D substrate rigidity using hydrogel scaffolds of photocrosslink-controllable stiffness. Specifically, our focus is on the role of membrane cholesterol and caveolae subdomains, and focal adhesion signaling in these responses. The responses of MSCs to substrate rigidity, particularly in a 3D context, represent an important regulatory mechanism of their biological activities, of particular relevance to their application in tissue engineering and regeneration. We postulate here that these responses involve integrin-mediated focal adhesion signaling, and are potentially sensitive to cellular cholesterol homeostasis. We therefore propose to analyze the effects of the cholesterol/Caveolin-1 (Cav-1)/caveolae membrane system, which is known to regulate focal adhesion signaling, on MSC substrate rigidity responses, with special attention to differentiation. Information on the effects of this system on MSC behavior may be important in a wider context, if it is affected in vivo by cholesterol-modifying drugs, which are widely clinically prescribed and thus may influence outcomes of regenerative therapies. We hypothesize that elevated cell membrane levels of cholesterol/Cav-1/caveolae decrease MSC sensitivity to substrate stiffness through increasing integrin endocytosis and decreasing focal adhesion signaling. To test our hypothesis, we propose three specific aims in which we will use cholesterol depletion, cholesterol supplementation, Cav-1 knockdown, and pharmacological inhibitors, to study the roles of MSC membrane cholesterol, Cav-1, caveolae, and focal adhesion signaling in MSC rigidity sensing in our 3D experimental platform. AIM 1: Test the effects of perturbations in cholesterol/Cav-1/caveolae homeostasis on MSC membrane properties and adhesive characteristics. This will verify that manipulation of cholesterol/Cav- 1/caveolae impacts aspects of the MSC cell membrane important to substrate sensing, including integrin expression, activation and internalization, and the strength of cell adhesion to defined substrates. AIM 2: Test the responses of MSCs with and without perturbations in cellular cholesterol/Cav-1/caveolae homeostasis to varied stiffness in a 3D context. This will determine how MSCs respond to varied substrate rigidity in 3D, in terms of their morphology, substrate adhesion, cytoskeletal organization, and differentiation, and if manipulation of cholesterol/Cav-1/caveolae affects these responses. AIM 3: Test the activity of integrin- activated focal adhesion signaling pathways in MSCs within soft and stiff 3D substrates, and the regulatory effects of cholesterol/Cav-1/caveolae on focal adhesion signaling and downstream differentiation as influenced by soft and stiff 3D scaffolds. This will determine if focal adhesion signaling is involved in MSC substrate rigidity responses, and if such involvement is affected by cholesterol/Cav-1/caveolae.

抽象的 我们提出了一项机械研究，对成年人类骨髓的生物学反应 间充质干细胞（MSC）至3D底物刚性，使用可控制的光蛋白链接控制的水凝胶支架 刚性。具体而言，我们的重点是膜胆固醇和小窝子域的作用，以及焦点 这些响应中的粘附信号传导。 MSC对底物刚性的响应，特别是在3D中 背景，代表其生物学活动的重要调节机制，特别相关 它们在组织工程和再生中的应用。我们在这里假设这些响应涉及 整联蛋白介导的局灶性粘附信号传导，并可能对细胞胆固醇稳态敏感。 因此，我们建议分析胆固醇/小窝1（CAV-1）/小窝膜系统的影响， 已知可以调节局部粘附信号传导，在MSC底物刚度响应上，并具有特殊 注意差异化。有关该系统对MSC行为影响的信息可能在A中很重要 更广泛的情况，如果它通过胆固醇改性药物在体内受到影响，这些药物在临床上被广泛处方，并且 因此，可能会影响再生疗法的结果。 我们假设胆固醇/CAV-1/Caveolae升高的细胞膜水平降低了MSC 通过增加整联蛋白内吞作用并减少局灶性，对底物刚度的敏感性 粘附信号传导。为了检验我们的假设，我们提出了三个具体目标，我们将使用胆固醇 耗尽，补充胆固醇，CAV-1敲低和药理抑制剂，以研究的作用 MSC膜胆固醇，CAV-1，小窝和局灶性粘附信号传导在我们的3D中的MSC刚度传感中 实验平台。目标1：测试胆固醇/cav-1/caveolae稳态中扰动的影响 MSC膜特性和粘合特性。这将验证是否操纵胆固醇/Cav- 1/caveolae影响MSC细胞膜的各个方面对底物感应很重要，包括整合素 表达，激活和内在化以及细胞粘附对定义底物的强度。目标2：测试 在细胞胆固醇/cav-1/caveolae稳态中具有和不扰动的MSC的反应 在3D上下文中刚度变化。这将确定MSC在3D中如何应对各种底物刚度的响应 其形态，底物粘附，细胞骨架组织和分化的术语，以及 胆固醇/CAV-1/Caveolae的操纵会影响这些反应。目标3：测试整联蛋白的活性 - 软和硬质3D底物内的MSC中激活的局灶性粘附信号通路，调节 受影响 通过柔软和僵硬的3D脚手架。这将确定MSC底物是否涉及焦点粘附信号传导 刚性反应，如果这种参与受到胆固醇/CAV-1/Caveolae的影响。